This proposal is focused on developing innovative new cryopreservation techniques employing ice-free preservation methods that will lead to the ability to bank complex vascularized tissue and organ grafts. Banking of large or complex tissues, such as limbs, and organs using current tissue banking practices employing conventional cryopreservation by freezing is not possible due to the well known damage caused by intra and extra-cellular ice formation. The company team has developed vitrification, cryopreserved storage in a ?glassy? rather than crystalline ice phase, for banking of small tissue samples including blood vessels, heart valve tissues and cartilage. However, scale up to larger tissues, more complex tissues and organs needs better ice control during rewarming with less risk of cytotoxicity. Our collaborators, Professor Duman, University of Notre Dame, and Professor Ben, University of Ottawa, have developed natural and synthetic antifreeze compounds respectively that demonstrate benefits in frozen cell systems. Duman has developed recombinant insect- derived antifreeze proteins and isolation methods for antifreeze glycolipids from several insects and a plant that are very effective at ice control. Ben has developed a chemical library that was originally based upon an antifreeze glycoprotein derived from a deep sea teleost. We will evaluate the best of these compounds during ice-free cryopreservation using a vascular tissue model to determine if they improve ice control during rewarming and reduce the risk of cryoprotectant toxicity by allowing lower cryoprotectant concentrations to be used. There are three specific aims in this Phase I feasibility study to identify potentially marketable methods to increase efficacy and minimize tissue damage during cryopreservation of complex natural and engineered tissues: In Specific Aim 1 we will prepare of test compounds, antifreeze glycolipids from natural sources and synthesize large quantities of synthetic antifreeze compounds. A stock of recombinant insect-derived antifreeze proteins is already available. In Specific Aim 2 we will evaluate the impact of antifreeze compounds individually upon ice-free cryopreserved blood vessels and in Specific Aim 3 the best antifreeze supplements will be combined. Evaluations will include ice control during ice-free vitrification and tissue viability and function assays. The technology developed in this proposal will be equally effective for stasis, storage, of engineered tissue constructs as natural tissues. This work will lead to the development of banking, by us and other cryobiology groups, for internal organs, such as livers and hearts, vascularized composite allografts, such as larynx, trachea, abdominal wall, knee, skeletal muscles, facial tissues and legs, as well as improved methods for critical natural and engineered tissues such as skin, heart valves and blood vessels.